利福平缓解糖皮质激素诱导ONFH的早期软骨细胞凋亡的实验研究(1)
[摘要] 目的 探讨糖皮质激素(Glucocorticoids，GC)对软骨细胞的影响及利福平对其的保护作用。 方法 从SD大鼠的膝关节分离关节软骨细胞进行细胞培养并用不同浓度的地塞米松(Dexamethasone，Dex)进行治疗，按照随机对照原则分为三组：NS(Con)组、Dex组和Dex+Rif组，测量软骨细胞中的活性氧(Reactive oxygen species，ROS)水平和甲状旁腺激素(Parathyroid hormone，PTH)/甲状旁腺激素相关肽受体(Parathyroid hormone-related peptide receptor，PTH1R)的表达。进一步通过Western blot检测研究软骨细胞中凋亡相关蛋白的表达水平。 结果 GC处理后，软骨下区出现空洞软骨异常，破骨细胞活性增强，同时通过GC处理后ROS水平和PTH1R表达增加。在Dex+Rif组中，利福平保护了免受Dex诱导的软骨细胞凋亡。 结论 ROS产生和PTH1R表达减少可使利福平减轻Dex处理软骨细胞的凋亡，软骨细胞的大量凋亡可能是早期ONFH的特征性标志。
[关键词] 利福平;糖皮质激素;股骨头坏死;软骨细胞
[Abstract] Objective To investigate impacts of glucocorticoids(GC) on chondrocytes and protection effects of rifampicin. Methods Chondrocytes were isolated from the knee joint of SD rats for cell culture and were administered by dexamethasone(Dex) at different concentrations. According to the principle of random control， the cells were randomly divided into three groups： The NS(Con) group， the Dex group and the Dex+Rif group. The level of reactive oxygen species(ROS) and expressions of parathyroid hormone(PTH)/ type 1 PTH-related peptide receptor (PTH1R) in chondrocytes were determined. Expressions of apoptosis-related proteins in chondrocytes were determined by western blot. Results After the treatment of GC， abnormally eroded cavities were formed in subchondral bone， accompanied by elevated osteoclast activity， as well as increased levels of ROS and PTH1R in chondrocytes. Rif protected chondrocytes against Dex-induced cell apoptosis in the Dex+Rif group. Conclusion Rif repressed Dex-induced chondrocyte apoptosis via ROS generation and PTH1R down-regulation. A high amount of chondrocyte apoptosis could be a typical picture of early osteonecrosis of the femoral head(ONFH).
[Key words] Rifampicin; Glucocorticoids; Osteonecrosis of the femoral head; Chondrocytes
股骨頭坏死(Osteonecrosis of the femeral head，ONFH)经常导致股骨头的进行性塌陷变形[1]，而GC给药导致的激素性ONFH是非创伤性中最常见的危险因素，多发生于35～55岁的青壮年，男性多于女性[2]。软骨细胞的凋亡和坏死等异常情况通常被认为是ONFH的特征性标志之一。目前，ONFH的发病机制仍不清楚，是国内外骨坏死研究的热点[3]。在许多研究中已经报道了关于GC诱导软骨细胞的凋亡和坏死等异常情况对ONFH的影响[14]。Liu LH等[4]通过在GC诱导的ONFH中，发现在症状出现之前，可以诊断软骨坏死，尤其是通过磁共振成像(MRI)，但在早期阶段仍没有令人满意的治疗ONFH的方法。如果早期阶段的变化得到明确说明，治疗方法及效果将会更好。由于GC诱导的生长迟缓与软骨细胞的凋亡增加有关[5]，本研究在ROS水平和PTH/PTH1R与细胞凋亡有关的基础上，假设GC诱导的细胞凋亡与ROS水平升高和PTH1R表达有关，通过体外研究软骨细胞，现报道如下。
1 资料与方法
1.1 试剂和材料
SD大鼠18只、不同浓度Dex(10-7～10-4 M)利福平、10%胎牛血清(FBS)、抗生素DMEM/F12;培养基5×103细胞/孔、CCK-8(Dojindo Laboratory，Kumamoto，Japan)、PI/Hoechst混合物(Beyotime生物技术研究所，中国江苏)、10 μmol/L DCFH-DA、兔抗鼠Caspase-3抗体、PTH1R抗体、羊抗兔荧光抗体、DAPI;SDS-PAGE(10%聚丙烯酰胺凝胶)。, http://www.100md.com(谢冰勇 陈紫璇 刘扶摇)
[关键词] 利福平;糖皮质激素;股骨头坏死;软骨细胞
[Abstract] Objective To investigate impacts of glucocorticoids(GC) on chondrocytes and protection effects of rifampicin. Methods Chondrocytes were isolated from the knee joint of SD rats for cell culture and were administered by dexamethasone(Dex) at different concentrations. According to the principle of random control， the cells were randomly divided into three groups： The NS(Con) group， the Dex group and the Dex+Rif group. The level of reactive oxygen species(ROS) and expressions of parathyroid hormone(PTH)/ type 1 PTH-related peptide receptor (PTH1R) in chondrocytes were determined. Expressions of apoptosis-related proteins in chondrocytes were determined by western blot. Results After the treatment of GC， abnormally eroded cavities were formed in subchondral bone， accompanied by elevated osteoclast activity， as well as increased levels of ROS and PTH1R in chondrocytes. Rif protected chondrocytes against Dex-induced cell apoptosis in the Dex+Rif group. Conclusion Rif repressed Dex-induced chondrocyte apoptosis via ROS generation and PTH1R down-regulation. A high amount of chondrocyte apoptosis could be a typical picture of early osteonecrosis of the femoral head(ONFH).
[Key words] Rifampicin; Glucocorticoids; Osteonecrosis of the femoral head; Chondrocytes
股骨頭坏死(Osteonecrosis of the femeral head，ONFH)经常导致股骨头的进行性塌陷变形[1]，而GC给药导致的激素性ONFH是非创伤性中最常见的危险因素，多发生于35～55岁的青壮年，男性多于女性[2]。软骨细胞的凋亡和坏死等异常情况通常被认为是ONFH的特征性标志之一。目前，ONFH的发病机制仍不清楚，是国内外骨坏死研究的热点[3]。在许多研究中已经报道了关于GC诱导软骨细胞的凋亡和坏死等异常情况对ONFH的影响[14]。Liu LH等[4]通过在GC诱导的ONFH中，发现在症状出现之前，可以诊断软骨坏死，尤其是通过磁共振成像(MRI)，但在早期阶段仍没有令人满意的治疗ONFH的方法。如果早期阶段的变化得到明确说明，治疗方法及效果将会更好。由于GC诱导的生长迟缓与软骨细胞的凋亡增加有关[5]，本研究在ROS水平和PTH/PTH1R与细胞凋亡有关的基础上，假设GC诱导的细胞凋亡与ROS水平升高和PTH1R表达有关，通过体外研究软骨细胞，现报道如下。
1 资料与方法
1.1 试剂和材料
SD大鼠18只、不同浓度Dex(10-7～10-4 M)利福平、10%胎牛血清(FBS)、抗生素DMEM/F12;培养基5×103细胞/孔、CCK-8(Dojindo Laboratory，Kumamoto，Japan)、PI/Hoechst混合物(Beyotime生物技术研究所，中国江苏)、10 μmol/L DCFH-DA、兔抗鼠Caspase-3抗体、PTH1R抗体、羊抗兔荧光抗体、DAPI;SDS-PAGE(10%聚丙烯酰胺凝胶)。, http://www.100md.com(谢冰勇 陈紫璇 刘扶摇)